Protocol #05
LABORATORY OF MOLECULAR PATHOLOGY
WGS library preparation
Using NEBNext Ultra IIFS DNA Library Prep Kit for lllumina Prep:
Fragmentation and end-repair
□ 00 Thaw all the reagents. Quick vortex and quick spin. Keep them on ice. Bring magnetic beads to room temperature, at least 30°C prior usage. Prepare fresh 80% ethanol. You will need 0.5 ml per sample.
□ 01 Check the Ultra II FS Reaction Buffer, make sure there is no precipitation.
□ 02 Vortex the Ultra II FS Enzyme Mix for 5 seconds before using and keep it on ice.
□ 03 Dilute your DNA sample to a total of 26 u.L with water. Ensure at least 300 ng of starting material - purified genomic DNA.
□ 04 Combine the 26 ul of DNA with 7 u.L Reaction Buffer and 2 u.L of Enzyme Mix to have a total volume of 35 ul. Quick vortex and quick spin.
□ 05 Incubate at 37 °C for 17 minutes, followed by 30 minutes at 65 °C in a cycler with a heated lid to 75 °C. Hold at 4 °C afterwards.
Adaptor ligation
□ 06 Combine the 35 u.L reaction mixture with 30 u.L Ligation Master Mix.
Add 1 u.L Ligation Enhancer and 2.5 ul of lllumina Adaptor for a total volume of 68.5 ui. Ensure proper mixing. The ligation master mix is very viscous. Pipette accordingly.
□ 07 Incubate at 20 °C for 15 minutes.
□ 08 Add 3 u.L of USER Enzyme to the ligation mixture.
□ 09 Mix well and incubate at 37 °C for 15 minutes with heated lid at 50 °C.
Size selection
□ 10 Add 28.5 u.L of O.lx TE to the mixture for a total volume of 100 ul.
□ 11 Vortex the magnetic beads vigorously to properly resuspend them.
□ 12 Add 40 u.L of the magnetic beads to the sample for a total volume of 140 ul. Mix the sample properly - vortex on high and follow with a very quick spin.
□ 13 Incubate at room temperature for 5 minutes.
□ 14 Put the samples on the magnetic stand and incubate another 5 minutes to separate the beads from the supernatant.
□ 15 Transfer the supernatant into a new tube. Do not discard the supernatant. Discard the magnetic beads (those contain large DNA fragments).
Protocol #05 LABORATORY
OF MOLECULAR PATHOLOGY
□ 16 Add 20 u.L of fresh resuspended magnetic beads. Mix the sample properly.
□ 17 Incubate at room temperature for 5 minutes.
□ 18 Put the samples on the magnetic stand and incubate for another 5 minutes.
□ 19 Remove and discard the supernatant. Do not disturb or discard the beads.
□ 20 Add 200 u.L of fresh 80% ethanol. Incubate for 30 seconds. Remove the supernatant and do not disturb the beads. Keep the samples on the magnet.
□ 21 Repeat step 20 for a second wash - add 200 ul of ethanol, wait 30 seconds and remove the ethanol. Do not disturb the beads.
□ 22 Quickly spin the samples to collect residual ethanol. Put the samples on the magnet and remove the rest of the ethanol with a 10 ul pipette.
□ 23 Air dry the beads if necessary. Do not overdry the beads. The beads need to be dark brown and glossy. When the beads turn light brown and start to crack, it is too late.
□ 24 Remove the samples from the magnet and add 17 u.L of O.lx TE buffer.
□ 25 Mix well-vortex followed by a quick spin.
□ 26 Incubate at RT for 5 minutes. Off the magnet.
□ 27 Put the samples on the magnet and incubate them for at least 2 minutes.
□ 28 Transfer 15 u.L of the supernatant to a new tube. Do not disturb the beads.
PCR enrichment
□ 29 Combine the PCR reaction: 15 u.L of the sample, 25 u.L of Q5 Master Mix and 5 u.L of i7 primer and 5 ul of i5 primer for a total reaction volume of 50 ul. Quick vortex and quick spin.
The 17 and i5 primers identify the sample. The combination of 15 and 17 must be unique for every sample. Watch out for barcode overlap with other samples and libraries.
□ 30 Run the following PCR programme:
Amplification programme	Temperature	Time
Initial denaturation	98 °C	30 seconds
		
Denaturation	98 °C	10 s fP- -
Annealing+Extension	65 °C	oX 75 s
		
Final extension	65 °C	5 min
	4°C	Hold
Protocol #05
LABORATORY OF MOLECULAR PATHOLOGY
PCR cleanup
□ 31 Vortex the magnetic beads to resuspend them.
□ 32 Add 45 u.L beads to the 50 ul of the PCR mixture for a total volume of 95 ul
□ 33 Incubate at room temperature for 5 minutes.
□ 34 Put the samples on the magnetic stand and incubate for another 5 minutes.
□ 35 Remove and discard the supernatant. Do not disturb or discard the beads.
□ 36 Add 200 u.L of fresh 80% ethanol. Incubate for 30 seconds. Remove the supernatant and do not disturb the beads. Keep the samples on the magnet.
□ 37 Repeat step 20 for a second wash - add 200 ul of ethanol, wait 30 seconds and remove the ethanol. Do not disturb the beads.
□ 38 Quickly spin the samples to collect residual ethanol. Put the samples on the magnet and remove the rest of the ethanol with a 10 ul pipette.
□ 39 Air dry the beads if necessary. Do not overdry the beads.
□ 40 Remove the samples from the magnet and add 33 u.L of O.lx TE buffer.
□ 41 Mix well-vortex followed by a quick spin.
□ 42 Incubate at RT for 5 minutes. Off the magnet.
□ 43 Put the samples on the magnet and incubate them for at least 2 minutes.
□ 44 Transfer 30 u.L of the supernatant to a new tube. Do not disturb the beads.
□ 45 Measure the concentration using Qubit.
□ 46 Asses library quality using FragmentAnalyzer.